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APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Dec. 1991, p. 3462-3469 Vol. 57, No. 12 0099-2240/91/123462-08$02.00/0 Copyright C) 1991, American Society for Microbiology Indigenous and Enhanced Mineralization of Pyrene, Benzo[a]pyrene, and Carbazole in Soils ROBERT J. GROSSER,1 DAVID WARSHAWSKY,2 AND J. ROBIE VESTAL'* Department of Biological Sciences, University of Cincinnati, Cincinnati, Ohio 452211 and Department of Environmental Health, University of Cincinnati, Cincinnati, Ohio 452672 Received 11 June 1991/Accepted 20 September 1991 We studied the mineralization of pyrene, carbazole, and benzo[a]pyrene in soils obtained from three abandoned coal gasification plants in southern Illinois. The soils had different histories of past exposure to hydrocarbon contamination and different amounts of total organic carbon, microbial biomass, and microbial activity. Mineralization was measured by using serum bottle radiorespirometry. The levels of indigenous mineralization of 14C-labeled compounds ranged from 10 to 48% for pyrene, from undetectable to 46% for carbazole, and from undetectable to 25% for benzo[a]pyrene following long-term (>180-day) incubations. Pyrene and carbazole were degraded with short or no lag periods in all soils, but benzo[a]pyrene mineralization occurred after a 28-day lag period. Mineralization was not dependent on high levels of microbial biomass and activity in the soils. Bacterial cultures that were capable of degrading pyrene and carbazole were isolated by enrichment, grown in pure culture, and reintroduced into soils. Reintroduction of a pyrene-degrading bacterium enhanced mineralization to a level of 55% within 2 days, compared with a level of 1% for the indigenous population. The carbazole degrader enhanced mineralization to a level of 45% after 7 days in a soil that showed little indigenous carbazole mineralization. The pyrene and carbazole degraders which we isolated were identified as a Mycobacterium sp. and a Xanthamonas sp., respectively. Our results indicated that mineralization of aromatic hydrocarbons can be significantly enhanced by reintroducing isolated polycyclic aromatic hydrocarbon-degrading bacteria. Concern about the presence of priority pollutants, such as polycyclic aromatic hydrocarbons (PAHs) and N-heterocy- clic aromatic compounds (NHAs), in the environment has increased as more is discovered about the potential carcino- genicity and microbial recalcitrance of these compounds. PAH degradation has been studied in the environment, but little is known about NHA degradation by microbial systems in the environment. These compounds enter the environ- ment in many ways, including combustion and accidental spilling of hydrocarbons and oils, as well as heat and power generation, refuse burning, fallout from urban pollution, and coal liquefaction and gasification processes (4). Microbiological degradation and transformation are thought to be the principal processes that result in the removal of PAHs and NHAs from the environment (35). Workers have isolated a wide variety of eukaryotic and prokaryotic organisms that have the ability to metabolize specific PAHs and NHAs (5, 7, 8, 11, 19, 39). However, even though PAHs containing more than three fused benzene rings are biodegraded in the environment, the isolation of micoorganisms that possess the required catabolic enzymes has been difficult. Four- and five-ring PAHs are considered to be recalcitrant molecules because of the resonance ener- gies of their structures and their low water solubilities (21). This is consistent with the results of studies that have shown that biodegradation rates decrease with increasing numbers of fused benzene rings in molecules (13). Recent studies have shown that bioremediation can have an overall positive effect on the removal of many PAH and NHA residues in soils (26, 38), soil-sediment slurries (27), and groundwater (28). The main objectives of this study were to determine the * Corresponding author. rates and extents of biodegradation of pyrene, carbazole, and benzo[a]pyrene (BaP) in impacted soil systems and to isolate microorganisms that are capable of degrading these compounds. The organisms which we isolated were tested for their influence on degradation of these compounds after the organisms were reintroduced into soils, and the effects of inoculum size on mineralization of the compounds were determined. MATERIALS AND METHODS Soils were obtained from three abandoned coal gasifica- tion plants in southern Illinois in June 1988. The samples were obtained from the top 10 cm of soil and were placed in sterile Nalgene containers (4 liters) for transport, and they were stored at 4°C until they were used. Two samples were collected from each plant site; one sample was a sample of an experimental soil near the contaminated area, and one sample was a sample of a control soil that was located at least 20 m away from the contaminated area. The plant at site 1 operated until 1945 and contained a large pit of tar waste; samples were taken from along the edge of the pit. Site 2 was an Environmental Protection Agency Superfund site that was undergoing remediation; the sample was obtained from a large depression formed by the removal of a gas storage tank and the surrounding soil. The processing plant at site 3 operated from the 1860s through the 1940s, and the sample was taken from an area that was about 6 m from the remnants of a gas holding tank. Although the soils at each site were visually contaminated with oils and hydrocarbons, areas of vegetation were common. All soil manipulations were performed under gold lights to prevent photooxidation (Armalite diffuser shields; Thermo- plastics, Sterling, N.J.). All soils were air dried at room temperature, passed through a no. 10 (2.00-mm) sieve, 3462 on March 25, 2020 by guest http://aem.asm.org/ Downloaded from

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APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Dec. 1991, p. 3462-3469 Vol. 57, No. 120099-2240/91/123462-08$02.00/0Copyright C) 1991, American Society for Microbiology

Indigenous and Enhanced Mineralization of Pyrene,Benzo[a]pyrene, and Carbazole in Soils

ROBERT J. GROSSER,1 DAVID WARSHAWSKY,2 AND J. ROBIE VESTAL'*Department of Biological Sciences, University of Cincinnati, Cincinnati, Ohio 452211 andDepartment of Environmental Health, University of Cincinnati, Cincinnati, Ohio 452672

Received 11 June 1991/Accepted 20 September 1991

We studied the mineralization of pyrene, carbazole, and benzo[a]pyrene in soils obtained from threeabandoned coal gasification plants in southern Illinois. The soils had different histories of past exposure tohydrocarbon contamination and different amounts of total organic carbon, microbial biomass, and microbialactivity. Mineralization was measured by using serum bottle radiorespirometry. The levels of indigenousmineralization of 14C-labeled compounds ranged from 10 to 48% for pyrene, from undetectable to 46% forcarbazole, and from undetectable to 25% for benzo[a]pyrene following long-term (>180-day) incubations.Pyrene and carbazole were degraded with short or no lag periods in all soils, but benzo[a]pyrene mineralizationoccurred after a 28-day lag period. Mineralization was not dependent on high levels of microbial biomass andactivity in the soils. Bacterial cultures that were capable of degrading pyrene and carbazole were isolated byenrichment, grown in pure culture, and reintroduced into soils. Reintroduction of a pyrene-degradingbacterium enhanced mineralization to a level of 55% within 2 days, compared with a level of 1% for theindigenous population. The carbazole degrader enhanced mineralization to a level of 45% after 7 days in a soilthat showed little indigenous carbazole mineralization. The pyrene and carbazole degraders which we isolatedwere identified as a Mycobacterium sp. and a Xanthamonas sp., respectively. Our results indicated thatmineralization of aromatic hydrocarbons can be significantly enhanced by reintroducing isolated polycyclicaromatic hydrocarbon-degrading bacteria.

Concern about the presence of priority pollutants, such aspolycyclic aromatic hydrocarbons (PAHs) and N-heterocy-clic aromatic compounds (NHAs), in the environment hasincreased as more is discovered about the potential carcino-genicity and microbial recalcitrance of these compounds.PAH degradation has been studied in the environment, butlittle is known about NHA degradation by microbial systemsin the environment. These compounds enter the environ-ment in many ways, including combustion and accidentalspilling of hydrocarbons and oils, as well as heat and powergeneration, refuse burning, fallout from urban pollution, andcoal liquefaction and gasification processes (4).

Microbiological degradation and transformation arethought to be the principal processes that result in theremoval of PAHs and NHAs from the environment (35).Workers have isolated a wide variety of eukaryotic andprokaryotic organisms that have the ability to metabolizespecific PAHs and NHAs (5, 7, 8, 11, 19, 39). However, eventhough PAHs containing more than three fused benzenerings are biodegraded in the environment, the isolation ofmicoorganisms that possess the required catabolic enzymeshas been difficult. Four- and five-ring PAHs are consideredto be recalcitrant molecules because of the resonance ener-gies of their structures and their low water solubilities (21).This is consistent with the results of studies that have shownthat biodegradation rates decrease with increasing numbersof fused benzene rings in molecules (13). Recent studieshave shown that bioremediation can have an overall positiveeffect on the removal of many PAH and NHA residues insoils (26, 38), soil-sediment slurries (27), and groundwater(28).The main objectives of this study were to determine the

* Corresponding author.

rates and extents of biodegradation of pyrene, carbazole,and benzo[a]pyrene (BaP) in impacted soil systems and toisolate microorganisms that are capable of degrading thesecompounds. The organisms which we isolated were testedfor their influence on degradation of these compounds afterthe organisms were reintroduced into soils, and the effects ofinoculum size on mineralization of the compounds weredetermined.

MATERIALS AND METHODS

Soils were obtained from three abandoned coal gasifica-tion plants in southern Illinois in June 1988. The sampleswere obtained from the top 10 cm of soil and were placed insterile Nalgene containers (4 liters) for transport, and theywere stored at 4°C until they were used. Two samples werecollected from each plant site; one sample was a sample ofan experimental soil near the contaminated area, and onesample was a sample of a control soil that was located atleast 20 m away from the contaminated area.The plant at site 1 operated until 1945 and contained a

large pit of tar waste; samples were taken from along theedge of the pit. Site 2 was an Environmental ProtectionAgency Superfund site that was undergoing remediation; thesample was obtained from a large depression formed by theremoval of a gas storage tank and the surrounding soil. Theprocessing plant at site 3 operated from the 1860s throughthe 1940s, and the sample was taken from an area that wasabout 6 m from the remnants of a gas holding tank. Althoughthe soils at each site were visually contaminated with oilsand hydrocarbons, areas of vegetation were common.

All soil manipulations were performed under gold lights toprevent photooxidation (Armalite diffuser shields; Thermo-plastics, Sterling, N.J.). All soils were air dried at roomtemperature, passed through a no. 10 (2.00-mm) sieve,

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MINERALIZATION OF PAHs AND NHAs IN SOIL 3463

placed in plastic bags, and stored at 4°C until they were used.Site 2 experimental soil had to be pulverized prior to usebecause it had a rocklike texture when it was dried.

Chemicals. Some of the 14C-labeled compounds which weused, their specific activities (in microcuries per millimole),and their suppliers were as follows: [4,5,9,10-14C]pyrene,40.0 to 60.0, Chemsyn Scientific, Lenexa, Kans.; [U-14C]car-bazole, 7.9, Sigma Chemical Co., St. Louis, Mo.; [7,10-14C]BaP, 52.0, Amersham Corp., Arlington Heights, Ill.; [9,10-14C]anthracene, 15.1, Amersham; [12-14C]benz[a]anthracene,49.0, Amersham; [1,4,5,9,10,13,13b,13c-14C]7H-dibenzo[c,g]carbazole, 2.0, Amersham; and [14-14C]dibenz[ajsacridine,13.8, Amersham. [U-14C]acetate was obtained from ICNBiochemicals, Inc., Costa Mesa, Calif., and had a specificactivity of 57 mCi mmol-1. The radiolabeled aromatic com-pounds were received in toluene, dried under a stream ofnitrogen, and resuspended in ethylene glycol monomethylether. For mineralization experiments, the 14C-labeled com-pounds were diluted in sterile deionized distilled water(DDW) before use. All other solvents and chemicals usedwere of reagent grade or better.

Media. Minimal basal salts (MBS) medium was made asdescribed by Skerman (36). MBS agar plates were made byadding 1.75% Noble agar (Difco). Yeast extract peptoneglucose broth (YEPG) contained (per liter) 0.2 g of yeastextract, 1.0 g of peptone, 2.0 g of glucose, and 0.2 g ofammonium nitrate. This broth was used as a multipurposegrowth medium. It was used at 1% strength in solid media forenumerating oligotrophic organisms in the soils and at 25%strength in solid media for isolating degrading organismsfollowing enrichment procedures. All preparations wereincubated at room temperature (22 to 24°C).

Soil biological analyses. Soil microbial biomass was deter-mined by using the lipid phosphate analysis method (40), asmodified by Findlay et al. (6). Soil microbial activity wasdetermined by measuring the rate of [14C]acetate incorpora-tion into microbial lipids (24), using 5% (wt/vol) soil slurries.Quadruplicate soil samples were incubated with 0.1 ml of[14C]acetate (0.200 ,uCi, 3.51 nmol) for 2 h. Duplicate abioticcontrols were prepared by adding the lipid extractants for atleast 30 min prior to the addition of [14C]acetate. Thenitrogen-dried lipid fraction was quantified by using a Pack-ard model 2200CA Tri Carb scintillation counter. Enumera-tion of soil microorganisms was accomplished by using themethods described by Wollum (41) and then plating thepreparation in triplicate onto 1% YEPG agar. All of theplates were counted after 2 weeks of incubation.

Soil chemical analysis. Soil pH was determined by usingthe method described by McLean (25). Soil total organiccarbon (TOC) content was determined gravimetrically aftercombustion at 550°C for 2 h by using triplicate 4-g soilsamples. Soil water-holding capacity (WHC) was deter-mined by using the methods described by Gardner (10).Hexane-soluble hydrocarbon content was determined grav-imetrically after extraction of 2 g of soil with 10 ml of hexanewith 1 h of shaking. Soil cation exchange capacity wasdetermined by using a modification of the method of Bas-comb (2) by exchanging BaCl2 with MgSO4. The unex-changed Mg remaining in solution was analyzed by induc-tively coupled plasma emission spectrometry with a LeemanPlasma spec 2.5 instrument.

Indigenous soil microorganism mineralization experiments.All of the mineralization experiments were performed byusing the serum bottle radiorespirometry method describedby Knaebel and Vestal (22) and quadruplicate 5-g samples ofsoil. Killed controls were prepared by autoclaving duplicate

samples at >121°C for at least 6 h on two consecutive dayswith cooling between treatments. The soils were wetted to80% WHC by adding sterile DDW and 0.1-ml aliquots ofprepared 14C-labeled compounds. The quantities of '4C-labeled compounds added were as follows: pyrene, 8.5 ngg-1 (0.002 ,uCi, 0.042 nmol); carbazole, 52.8 ng g-' (0.004,Ci, 0.316 nmol); and BaP, 84 ng g-' (0.003 ,uCi, 0.333nmol). The serum bottles were vortexed briefly to mix thelabeled compounds into the soils. The radioactivity presenton the wicks was quantified by liquid scintillation counting,using an external standard protocol.

Chemical analysis. After the indigenous mineralizationexperiments were completed, mass balance extractions wereperformed. Soils were extracted with 10 ml of sterile DDW,shaken overnight, and centrifuged at 12,100 x g for 10 min.The radioactivity associated with the supernatant was mea-sured. The soil pellet was then extracted with DDW-meth-ylene chloride (1:2) and homogenized with a Tissumizerinstrument (Tekmar, Inc., Cincinnati, Ohio) run at 70% ofthe maximum setting for 30 s. The samples were centrifugedat 48,200 x g for 10 min, and the methylene chloride phasewas transferred to 20-ml glass scintillation vials and driedunder nitrogen. Ethyl acetate (15 ml) was added to the soilsamples, the preparations were shaken for 24 h and centri-fuged, and the ethyl acetate phase was combined with thenitrogen-dried methylene chloride residue. Then the ethylacetate extraction procedure was repeated. The radioactiv-ity associated with the dried total organic extract layer wasquantified by liquid scintillation spectrometry. The remain-ing 10-ml aqueous layer was removed, and the radioactivitywas quantified. To determine the potential chemilumines-cence resulting from the soil extractions, soils that receivedno '4C were subjected to the same extraction procedure.Any radioactivity detected in these controls was subtractedfrom the sample levels. A quench curve was constructed byusing extracted organic material from the unspiked soils.The levels of nonextractable '4C residues remaining in thesoil following extraction were determined by combustion ofthree 0.5-g portions of each sample (10).

Isolation of degraders. Degraders were isolated by analogenrichment, using a continuous enrichment microcosm withsite 1 and site 3 experimental soils. Soil (20 g) was added to1 liter of MBS medium in a 2-liter Erlenmeyer flask withconstant aeration, and then selected PAH and NHA com-pounds in an organic carrier (usually acetone) were added.Enrichments were made with weekly additions of naphtha-lene, phenanthrene, and anthracene (50 mg liter-1). Pyreneand BaP (50 mg liter-') were added to these enrichmentsafter several weeks of exposure to the smaller compounds.After 4 weeks, aliquots (10 ml) were removed from eachenrichment microcosm and added to MBS medium forfurther selection; 50 mg of an individual PAH or NHA perliter was then added to these subenrichments. An increase inturbidity compared with controls (MBS medium containing50 mg of a compound per liter) was used as an indicator ofgrowth on the compound. After several transfers, aliquotswere plated onto 25% YEPG plates and sprayed with a 2%solution of the enrichment compound in an organic carrier(20). Presumptive degraders were detected by noting clearzones around isolated colonies following 2 to 4 weeks ofincubation.To confirm utilization of a compound, presumptive de-

graders were grown in MBS medium supplemented with thecompound and cofactors (if required) and then subjected tomineralization experiments. The pyrene-degrading bacte-rium was cultured in MBS medium containing the following

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3464 GROSSER ET AL.

TABLE 1. Chemical and microbiological characteristics of southern Illinois soils used in mineralization experimentsa

Sample TOC content Hexane-extractable WHC Cation exchange Biomass (mean nmol of b Plate counts (CFU/gpH (% of dr wt) hydrocarbon content M capacity lipid phosphate per g Activity [dry wt] of soil)'

Site Soil ( o ry w (mg/g[dry wt] of soil) (meq/100 g of soil) [dry wt] of soil)

1 Exptl 4.4 24 ± 0.4 6 0.9 71 ± 2.6 138 ± 4 29 ± 2 56 ± 5 2.0 x 106Control 4.5 7 ± 0.1 1 0.2 58 ± 0.9 131 ± 6 22 ± 2 111 ± 14 6.0 x 106

2 Exptl 7.8 2 ± 0.0 1 0.2 31 ± 0.9 77 ± 16 106 ± 6 4 ± 1 1.0 x 105Control 7.7 8 ± 0.2 0.4 0.4 56 ± 2.8 129 ± 30 124 ± 70 50 ± 3 2.3 x 107

3 Exptl 4.4 39 ± 0.6 20 0.8 38 ± 3.1 99 ± 24 37 ± 2 9 ± 2 1.7 x 106Control 7.7 12 ± 0.3 3 0.1 56 ± 3.4 121 ± 22 124 ± 24 58 ± 3 6.4 x 107

a Values are means ± standard deviations for three replicates.b Microbial activity was measured as the rate of [14C]acetate incorporation into lipids. The values are the disintegrations per minute per hour per nanomole of

lipid phosphate.c Determined on oligotrophic agar.

cofactors: 250 ,ug of yeast extract per liter, 250 ,ug of peptoneper liter, 250 ,ug of soluble starch per liter, and 0.5 ,ug ofpyrene per ml (14). The carbazole degrader was cultured inMBS medium containing 37.5 ,ug of carbazole per ml.Cultures were harvested by centrifugation and resuspendedin fresh MBS medium. The resuspended cells were dividedin half; one-half was used for live cell mineralization, and theother half was autoclaved to be used for determining theeffects of killed cell biomass on mineralization. The numbersof colony-forming units per milliliter were determined byinoculating live cells onto 25% YEPG plates. The controlsconsisted of MBS medium containing 14C-labeled com-pounds in sterile serum bottles. The samples were shakencontinuously and were sampled for 2 weeks.

Reintroduction of microorganisms into soil. Harvested,resuspended cells (viable or autoclaved) were added to 5-gsoil samples to bring the soil to 80% WHC, and autoclavedcontrol soils were brought to 80% WHC with sterile DDW.The 14C-labeled compounds (56 ng of carbazole per g of soil[0.002 ,uCi, 0.335 nmol] and 37.8 ng of pyrene per g of soil[0.002 ,uCi, 0.187 nmol]) were added in combination withresuspended cells or DDW. Additional experiments weredone to determine the effects of different concentrations ofreintroduced cells on mineralization.

RESULTS

Soil characterization. The soils varied widely in terms ofthe chemical and microbiological characteristics which we

DPM Recovered (%)

0 10 20 30 40 50 60 70Time (in Days)

FIG. 1. Indigenous mineralization of pyrene by six southernIllinois soils. Exp, experimental soil; Cont, control soil.

measured (Table 1). The pH and TOC content valuesshowed much variation among the soils, while the cationexchange capacities were similar. In general, the experimen-tal soils had higher TOC and hydrocarbon levels than thecontrol soils. The microbiological characteristics of the soilsshowed that there were higher microbial activity rates in thecontrol soils than in the experimental soils. The activitiesranged from twice as high for site 1 control soil to 12 times ashigh for site 2 control soil. The biomass measurementsshowed similar trends but not to the same extent. Theoligotrophic plate counts were also higher in the control soilsthan in the experimental soils.

Mineralization. We observed disparate levels of indige-nous mineralization in the different soils for the compoundswhich we tested (Fig. 1 through 3). (Data points after day 63were not included in the graphs for pyrene and carbazoleindigenous mineralization because the curves had becomeasymptotic. Incubations were continued for 184 days forpyrene and for 198 days for carbazole.) After 60 days, thelevels of indigenous mineralization of pyrene in the six soilsranged from 10 to 48% (Fig. 1). Only site 2 experimental soilexhibited a lag period before mineralization began. Thegreatest amount of mineralization occurred in site 2 experi-mental soil; the lowest amount occurred in site 3 control soil.The levels of indigenous carbazole mineralization variedfrom the background level to 46% after 60 days (Fig. 2). Nolag period was observed in these experiments. Site 2 exper-imental soil exhibited the highest mineralization rate andlevel, and site 1 control soil exhibited the lowest. Indigenous

0 10 20 30 40 50 60 70Time (in Days)

FIG. 2. Indigenous mineralization of carbazole by six southernIllinois soils. Exp, experimental soil; Cont, control soil.

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MINERALIZATION OF PAHs AND NHAs IN SOIL 3465

DPM Recovered (%)

100 150

Time (in Days)

FIG. 3. Indigenous mineralization of BaP by six southern Illinoissoils. Exp, experimental soil; Cont, control soil.

mineralization of BaP (Fig. 3) occurred after an extensive lagperiod, and the overall mineralization levels were very low.A measurable level of label recovery (25%) was observedonly with site 2 experimental soil following a 3- to 4-week lagperiod. The standard deviations for the data in these miner-alization experiments were less than 10% in all cases.

Mass balances. Tables 2 through 4 show the results ob-tained from mass balance extractions of added 14C-labeledcompounds from soils following indigenous mineralizationexperiments. In general, high percentages of all three com-

pounds were recovered from most soils. Little or no 14C_labeled compound was found in the aqueous soil extracts ofall soils. If a compound did not exhibit measurable levels ofmineralization, the highest levels of recovery of added 14Cwere found in the organic and combustion portions of theextracts. The level of BaP recovery was low in site 1experimental soil and site 3 experimental soil which con-tained high amounts of TOC. We found the highest levels ofrecovery of 14C-labeled compounds with carbazole; theselevels ranged from 71 to 114% in the test samples from thesix soils. In general, we observed lower levels of recoverywith killed controls than with test samples for all compoundsand all soils.Pure culture mineralization experiments. We found several

isolates that were presumptive carbazole and pyrene degrad-ers. For each compound, one isolate was selected for furtherstudy. In addition to the three principal compounds whichwe studied, anthracene, phenanthrene, benz[a]anthracene,and acridine were sprayed onto plates prepared from theoriginal enrichment microcosms to identify possible degrad-ers. We found many isolates that produced clearing ofanthracene and phenanthrene on spray plates, but no iso-lates on plates sprayed with acridine and benz[a]anthracene.An isolated pyrene-degrading bacterium in MBS medium

at a cell density of 9 x 106 CFU ml-' mineralized ca. 55% ofthe added ['4C]pyrene within 2 days. The carbazole degraderat a density of 108 cells per ml mineralized ca. 60% of theadded ["4C]carbazole after 2 days. Mineralization was notobserved in the controls (data not shown). This verified thatclearing on spray plates by these isolates was due to utiliza-tion of the compound and not to another process, such as

solubilization. Attempts to isolate a BaP-degrading microor-ganism were unsuccessful.

Reintroduction of isolates to soil. As Fig. 4 shows, pyrenemineralization was enhanced to a level of 58% within 1 weekafter we reintroduced the pyrene-degrading bacterium at aconcentration of 2 x 109 CFU g-1 into site 2 experimentalsoil. Enhancement of mineralization occurred in both activeand autoclaved soils. With the active soil containing auto-claved cell biomass we observed mineralization curvescomparable to those of the native soil. Autoclaved soilcontaining autoclaved cells showed no mineralization. Min-eralization was complete within the first 1 week when livecells were reintroduced into the soil, while the other jl'per-alizations proceeded at slower rates and exhibited short lagperiods. We found that reintroduction of three differentconcentrations of the pyrene-degrading bacterium had noeffect on the extent of pyrene mineralization during a 14-daystudy; the values obtained for the three concentrations were

ca. 55% for 107 CFU g-1, ca. 62% for 5 x 107 CFU g-1, andca. 58% for 108 CFU g-'. Autoclaved cells added to the soilproduced mineralization kinetics similar to those of theindigenous control.Almost one-half (48%) of the added [14C]carbazole was

recovered as 14CO2 from site 1 control soil 14 days after thereintroduction of 4 x 107 CFU of the carbazole degrader perg (Fig. 5). Very little carbazole mineralization was measuredin any of the other samples. Reintroduction of different

TABLE 2. Mass balance determinations for pyrene indigenous mineralization experiments in southern Illinois soils

Sample % Recovery ina:

Site Soil Treatment CO Aqueous Organic Combustion Recoveryphase phase phase

1 Exptl Live 44.2 ± 3.9 0.1 + 0.0 0.6 ± 1.3 13.0 + 1.7 58.0Autoclaved 2.1 ± 0.1 b 11.8 ± 1.6 12.9 ± 1.6 26.8

Control Live 35.0 ± 2.7 0.6 + 0.1 8.8 ± 1.3 17.3 ± 2.1 61.7Autoclaved 2.5 ± 0.1 0.1 ± 0.0 1.3 ± 1.5 2.1 ± 0.8 73.9

2 Exptl Live 51.7 + 2.1 0.4 ± 0.1 3.1 ± 0.3 11.3 ± 1.0 66.5Autoclaved 3.0 ± 0.2 0.04 ± 0.1 46.8 ± 1.1 10.0 ± 0.4 59.8

Control Live 17.3 ± 1.6 0.5 ± 0.1 2.8 ± 0.4 24.5 ± 2.7 45.0Autoclaved 1.9 ± 0.0 _b 16.5 ± 0.1 20.8 ± 2.8 39.2

3 Exptl Live 26.7 ± 4.8 0.4 ± 0.1 2.2 ± 1.3 7.0 ± 1.7 36.2Autoclaved 2.1 ± 0.0 0.1 ± 0.0 4.6 ± 2.1 1.8 ± 1.6 8.5

Control Live 15.8 ± 1.6 0.9 ± 0.2 10.2 ± 3.0 45.3 ± 1.9 72.2Autoclaved 2.9 ± 0.1 0.02 ± 0.0 49.3 + 1.2 38.4 ± 0.6 90.7

Values are mean ± standard deviation percentages of recovery of added '4C-labeled compound after the extraction procedure described in the text. For thelive soil samples four replicates were used, and for the autoclaved soil samples two replicates were used.

b The value obtained was less than the value obtained for the extracted unspiked soil sample.

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TABLE 3. Mass balance determinations for carbazole indigenous mineralization experiments in southern Illinois soils

Sample % Recovery ina:

Site Soil Treatment C02 Aqueous Organic Combustion Recoveryphase phase phase

1 Exptl Live 5.9 ± 1.3 0.4 ± 0.1 2.6 ± 2.7 99.2 ± 9.9 108.2Autoclaved 1.2 ± 0.0 __b 4.3 ± 0.7 54.8 ± 1.1 60.2

Control Live 4.9 ± 1.9 1.0 ± 0.1 17.6 ± 3.6 73.6 + 3.6 97.0Autoclaved 1.3 ± 0.1 0.8 ± 0.1 14.1 ± 0.5 98.5 ± 1.4 114.6

2 Exptl Live 51.0 ± 1.2 1.0 + 0.1 3.5 ± 0.6 38.8 ± 3.1 94.2Autoclaved 3.2 ± 0.3 3.3 ± 0.5 41.4 ± 1.6 33.6 ± 8.1 81.5

Control Live 37.9 ± 3.4 0.8 ± 0.1 0.9 ± 0.2 39.2 ± 1.4 78.7Autoclaved 1.3 ±0.1 0.2 + 0.1 16.2 ± 0.8 52.3 ± 1.0 69.9

3 Exptl Live 10.1 + 2.2 0.8 ± 0.1 2.7 ± 0.8 67.0 + 2.0 70.6Autoclaved 1.1 ± 0.1 0.1 ± 0.0 6.9 + 0.4 13.1 ± 1.3 21.2

Control Live 33.0 ± 2.2 1.0 ± 0.1 3.9 ± 0.8 42.0 ± 2.0 80.0Autoclaved 1.1 ± 0.1 0.2 + 0.0 36.2 ± 0.4 51.0 ± 1.3 88.6

a See Table 2, footnote a.b See Table 2, footnote b.

numbers of the carbazole degrader into soil showed that BaP (21%), while the carbazole degrader utilized carbazolecarbazole mineralization depended on the concentration of but none of the other NHAs or PAHs tested.reintroduced cells; the levels of mineralization for threeconcentrations were ca. 29% for 106 CFU g-1, ca. 38% for 5 DISCUSSIONx 106 CFU g-1, and ca. 42% for 107 CFU g-1 after 14 days.

Isolate characteristics. The pyrene degrader was tenta- We found that pyrene, carbazole, and BaP were degradedtively identified as a Mycobacterium species on the basis of in the soils used in this study. Following an enrichmentmorphology and the acid-fast and Gram reactions. A fatty procedure, we isolated microorganisms that mineralizedacid profile analysis (MIDI, Newark, Del.) of this isolate pyrene and carbazole. By growing these isolates in pureproduced no match with the fatty acid profiles present in the culture on the relevant compound and reintroducing themdata base. The colonies were round, smooth, convex, and into soil, it was possible to enhance mineralization of theyellow on 1% YEPG agar. The carbazole degrader was compounds.tentatively identified as Xanthomonas ampelina on the basis The control soils from each site exhibited higher levels ofof a fatty acid analysis. This isolate produced round, microbial activity and contained higher numbers of microor-smooth, raised, colonies that were beige to white on 1% ganisms and the same or greater levels of biomass than theYEPG agar. experimental soils. However, microbial population numbersWe analyzed mineralization of other PAHs and NHAs by and levels of activity are not necessarily reliable indicators

the pyrene- and carbazole-degrading isolates in liquid cul- of the potential for degradation of recalcitrant molecules byture following growth on their respective compounds. The microbes (16, 34). Herbes (16) found that rate constants fororganisms were given the following 14C-labeled com- anthracene transformation were not related to total copi-pounds: anthracene, carbazole, pyrene, benz[a]anthracene, otrophic numbers (microbial activity and biomass) in sedi-BaP, dibenz[ajjacridine, and dibenzo[c,g]carbazole. The ments in the vicinity of a coal coking wastewater dischargepyrene-degrading isolate mineralized carbazole (6%) and but proposed that continuous inputs of PAHs produced an

TABLE 4. Mass balance determinations for BaP indigenous mineralization experiments in southern Illinois soils

Sample % Recovery ina:

Sitk Soil Treatment C02 Aqueous Organic Combustion Recoveryphase phase phase

1 Exptl Live 3.4 ± 0.4 0.2 ± 0.1 5.1 ± 5.1 27.1 ± 3.0 35.8Autoclaved 1.8 ± 0.0 b 9.9 ± 1.1 20.5 ± 1.0 32.1

Control Live 2.6 ± 0.2 0.2 ± 0.1 39.9 ± 2.9 40.6 ± 1.0 83.3Autoclaved 2.1 ± 0.1 0.02 ± 0.0 38.2 ± 1.2 45.2 ± 5.6 85.5

2 Exptl Live 25.3 ± 5.7 0.5 + 0.1 21.7 ± 3.3 39.4 ± 4.5 86.8Autoclaved 1.7 ± 0.0 b 66.3 ± 0.1 20.6 ± 0.6 88.7

Control Live 6.6 ± 0.6 0.4 ± 0.3 15.0 ± 1.3 55.0 ± 3.6 77.0Autoclaved 1.5 ± 0.0 b 22.3 ± 0.6 52.1 ± 1.0 75.9

3 Exptl Live 2.8 ± 0.3 0.1 ± 0.1 7.1 ± 1.1 6.3 ± 1.3 16.3Autoclaved 1.5 ± 0.1 0.1 ± 0.0 8.4 ± 1.0 5.4 ± 1.3 15.4

Control Live 4.4 ± 0.1 0.6 ± 0.2 24.3 ± 3.3 63.5 ± 13 92.8Autoclaved 1.6 ± 0.0 0.02 ± 0.1 33.6 ± 1.0 47.6 ± 0.5 82.7

See Table 2, footnote a.b See Table 2, footnote b.

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% DPM Recovered

20 30Time (in days)

FIG. 4. Enhanced mineralization of pyrersoil by reintroduction of pyrene-degrading Iheat treatment.

increased ability within the microbial (certain PAHs. This phenomenon was <study; we found that site 2 experimentzmicrobial activity of all soils yet had thtion potential with the compounds stithat additional factors, such as bioavaand selection for degrading populationsthe breakdown of these compounds.The molecular structure and size c

pound in combination with soil charactioverall mineralization kinetics of a pStudies of PAH degradation in the envithat degradation rates are inversely relafused benzene rings (5). Our findingsrelationship, as pyrene was mineralizeiand at a faster rate than BaP. Pyrene Msoils in this study even though pyrenePAHs are considered to be fairly recaloThe three compounds produced diffi

alization curves for indigenous mineralPark et al. (30) expressed the degrcompounds in two soils by using on

model. The NONLIN module of SYSTAT (Systat, Evan-ston, Ill.) was used to describe the kinetics observed for eachof the indigenous mineralization curves in this current study,and the best-fit model was determined by using the methodof Robinson (32) (data not shown). The best-fit model oftendid not resemble the observed mineralization curve, suggest-ing that available mineralization models do not adequatelydescribe the processes involved in environmental PAH andNHA uptake and metabolism. For this reason, the kinetic

- Soil analysis was not continued.- Soil * Cells The pyrene- and carbazole-degrading isolates showed

Soil * HT Cells limited substrate specificity. The pyrene degrader mineral-} HT Soil * Cells ized BaP, which is similar in structure to pyrene (with thesHT Soil * HT Cells _ addition of a fifth benzene ring), suggesting that the enzy-

matic breakdown of BaP and the enzymatic breakdown of40 so pyrene by this organism may be similar. Rogoff (33) sug-

gested that the more similar PAHs are structurally, the morene in site 2 experimental likely the oxygenases for initial hydroxylation are also)actenia. HT, autoclave similar. Heitkamp and Cerniglia (14) noted that their pyrene-

degrading isolate degraded the four-ring PAHs fluorantheneand pyrene more rapidly than naphthalene and phenan-threne. The three-ring PAH anthracene was not utilized by

community to utilize the pyrene-degrading microorganism. No mineralization ofalso observed in this BaP was observed with the Mycobacterium sp. isolated inal soil had the lowest the study of Heitkamp and Cerniglia, but 25% of the totalie highest mineraliza- BaP residues were detected as metabolites. The pyrene-udied. This suggests degrading bacterium isolated in our study mineralized BaPtilability of substrate (21%) and will require further investigation., may be involved in The carbazole-degrading isolate did not mineralize the

other 14C-labeled substrates tested, which is consistent with)f an aromatic com- the findings of Finnerty et al. (7), whose carbazole-degradingeristics can affect the isolates degraded other NHA compounds that were similariarticular compound. in molecular size to carbazole but did not utilize PAHs. Ourronment have shown isolate did not utilize the five-ring NHAs dibenz[ajfacridineLted to the number of and dibenzo[c,g]carbazole, suggesting that breakdown ofsupport this inverse these larger compounds may be affected by their bioavail-d to a greater extent ability or may require induction of different enzymes. Thisvas mineralized in all organism may also possess enzymes which have narrowand other four-ring specificities.

citrant. The concentrations of cells used for the reintroductionerent types of miner- experiments affected the levels of mineralization of carba-lization experiments. zole, but had no affect on pyrene mineralization. This findingadation of 14 PAH is similar to the observations of Heitkamp and Cerniglia (15),Lly a first-order rate who found that doubling the inoculum size of their pyrene-

degrading Mycobacterium sp. increased the degradation ofpyrene only slightly in an aquatic sediment. Bacterial sur-vival in soil is difficult to measure and can be affected bycompetition with indigenous bacteria, as well as competition

- Soil for nutrients. The numbers of cells added per gram soil in- Soil + Cells this work (106 to 108 cells per g of soil) were similar to theL Soil * HT Cells numbers added in other reintroduction studies (29, 31, 37).HT Soil * Celia In these studies workers observed variable levels of survivalHT Soil + HT Cells of reintroduced bacterial populations.

The TOC present in the soils may have played a role in theavailability of the compounds to the organisms. The amountof TOC present is known to affect the adsorption anddesorption of many hydrophobic compounds in soils (1, 18).The partition coefficients (Kp) of pyrene and other hydrocar-bons exhibited linear relationships with the amounts oforganic carbon in sediments, and the differences in sorptionwithin silt and clay fractions were found to be caused by

40 50 60 differences in organic carbon content (23). Herbes andSchwall (17) suggested that sorptive immobilization of larger

bazole in site 1 control PAH compounds on sediment particles limited bioavailabil-ing bacteria. HT, auto- ity to microorganisms. The lack of BaP degradation in

sediment was explained by partitioning of the compound

0 10 20 30Time (in days)

FIG. 5. Enhanced mineralization of car]soil by reintroduction of carbazole-degradiclave heat treatment.

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3468 GROSSER ET AL.

onto sediment particles, leaving only a small amount avail-able for uptake and transformation by microbes (16). In thisstudy, site 2 experimental soil had the lowest organic carboncontent and exhibited the highest mineralization levels forthe three compounds studied.The effects of TOC on mass balance are obvious if Table

1 is compared with Tables 2 through 4. Soils with high TOCcontents (site 1 experimental soil and site 3 experimentalsoil) showed lower levels of recovery of the added 14C-labeled compounds. Many workers have examined the sorp-tion of PAH compounds and found varying degrees ofrecovery from soil and other environmental matrices byusing many methods and extractants (3, 9, 12). The extrac-tion protocol used in this study was selected following ananalysis of the methods available and was devised forpracticality (rapid analysis of many samples) and ease ofreplication. The choice of organic extracts and the length ofextract time used in this study may not have allowedcomplete partitioning of the compounds from soils contain-ing high levels of organic carbon.

Interest is growing in the use of natural microbial popula-tions to enhance cleanup of chemical pollution in the envi-ronment. The future success of bioremediation depends ondetermining the ability of indigenous soil microbial popula-tions to biodegrade pollutants, as well as isolation andfurther characterization of microorganisms that possess theability to degrade polluting chemicals.

ACKNOWLEDGMENTS

This work was supported by grant P42 ES4908 from the NationalInstitute of Environmental Health Sciences.We thank K. Haws, D. Moll, S. Sutton, D. Knaebel, and C.

Johnston for helpful discussions. We also thank D. Feldhake forhelp in beginning the experiments and J. Caruso and the Universityof Cincinnati Department of Chemistry for the use of the inductivelycoupled plasma emission spectrometry instrument.

REFERENCES1. Abdul, A. S., and T. L. Gibson. 1986. Equilibrium batch

experiments with six polycyclic aromatic hydrocarbons and twoaquifer materials. Hazard. Waste Hazard. Mater. 3:125-137.

2. Bascomb, C. L. 1964. Rapid method for the determination ofcation-exchange capacity of calcareous and non-calcareoussoils. J. Sci. Food Agric. 15:821-823.

3. Bulman, T. L., S. Lesage, P. J. Fowlie, and M. D. Webber. 1985.The persistance of polynuclear aromatic hydrocarbons in soil.PACE Report 85-2. Environmental Protection Service, Waste-water Technology Centre, Burlington, Ontario, Canada.

4. Cerniglia, C. E. 1984. Microbial metabolism of polycyclic aro-matic hydrocarbons. Adv. Appl. Microbiol. 30:31-71.

5. Cerniglia, C. E., and M. A. Heitkamp. 1987. Microbial degra-dation of polycyclic aromatic hydrocarbons in the aquaticenvironment, p. 41-68. In U. Varanasi (ed.), Metabolism ofpolycyclic aromatic hydrocarbons in the aquatic environment.CRC Press, Inc., Boca Raton, Fla.

6. Findlay, R. H., G. M. King, and L. Watling. 1989. Efficacy ofphospholipid analysis in determining microbial biomass in sed-iments. Appl. Environ. Microbiol. 55:2888-2893.

7. Finnerty, W. R., K. Shockley, and H. Attaway. 1983. Microbialdesulfurization and denitrogenation of hydrocarbons, p. 83-91.In J. E. Zajic, D. C. Cooper, T. R. Jack, and N. Kosaric (ed.),Microbial enhanced oil recovery. PennWell Books, Tulsa, Okla.

8. Foght, J. M., and D. W. S. Westlake. 1988. Degradation ofpolycyclic aromatic hydrocarbons and aromatic heterocycles bya Pseudomonas species. Can. J. Microbiol. 34:1135-1141.

9. Fowlie, P. J., and T. L. Bulman. 1986. Extraction of anthraceneand benzo(a)pyrene from soil. Anal. Chem. 58:721-723.

10. Gardner, W. H. 1982. Water content, p. 493-544. In A. Klute(ed.), Methods of soil analysis, part 1. Physical and mineralog-

ical methods. American Society of Agronomy, Inc., Soil Sci-ence Society of America, Inc., Madison, Wis.

11. Gibson, D. T., V. Mahadevan, D. M. Jerina, H. Yagi, andH. J. C. Yeh. 1975. Oxidation of the carcinogens benzo(a)py-rene and benz(a)anthracene to dihydrodiols by a bacterium.Science 189:295-297.

12. Griest, W. H., J. E. Caton, M. R. Guerin, L. B. Yeatts, andC. E. Higgins. 1973. Extraction and recovery of polycyclicaromatic hydrocarbons from highly sorptive matrices such as flyash, p. 819-828. In A. Bjorseth and A. J. Dennis (ed.), Polynu-clear aromatic hydrocarbons: chemistry and biological effects.4th International Symposium. Battelle Press, Columbus, Ohio.

13. Heitkamp, M. A., and C. E. Cerniglia. 1987. The effects ofchemical structure and exposure on the microbial degradation ofpolycyclic aromatic hydrocarbons in freshwater and estuarineecosystems. Environ. Toxicol. Chem. 6:535-546.

14. Heitkamp, M. A., and C. E. Cerniglia. 1988. Mineralization ofpolycyclic aromatic hydrocarbons by a bacterium isolated fromsediment below an oil field. Appl. Environ. Microbiol. 54:1612-1614.

15. Heitkamp, M. A., and C. E. Cerniglia. 1989. Polycyclic aromatichydrocarbon degradation by a Mycobacterium sp. in micro-cosms containing sediment and water from a pristine ecosys-tem. Appl. Environ. Microbiol. 55:1968-1973.

16. Herbes, S. E. 1981. Rates of microbial transformation of poly-cyclic aromatic hydrocarbons in water and sediments in thevicinity of a coal-coking wastewater discharge. Appl. Environ.Microbiol. 41:20-28.

17. Herbes, S. E., and L. R. Schwall. 1978. Microbial transformationof polycyclic aromatic hydrocarbons in pristine and petroleum-contaminated sediments. Appl. Environ. Microbiol. 35:306-316.

18. Karickhoff, S. W., D. S. Brown, and T. A. Scott. 1979. Sorptionof hydrophobic pollutants on natural sediments. Water Res.13:241-248.

19. Kiyohara, H., and K. Nagao. 1978. The catabolism of phenan-threne and naphthalene by bacteria. J. Gen. Microbiol. 105:69-75.

20. Kiyohara, H., K. Nagao, and K. Yana. 1982. Rapid screen forbacteria degrading water-insoluble, solid hydrocarbons on agarplates. Appl. Environ. Microbiol. 43:454-457.

21. Klevens, H. B. 1950. Solubilization of polycyclic hydrocarbons.J. Phys. Colloid Chem. 54:283-298.

22. Knaebel, D. B., and J. R. Vestal. 1988. A comparison of doublevial to serum bottle radiorespirometry to measure microbialmineralization in soils. J. Microbiol. Methods 7:309-317.

23. Lambert, S. M. 1967. Functional relationship between sorption insoil and chemical structure. J. Agric. Food Chem. 15:572-576.

24. McKinley, V. L., T. W. Federle, and J. R. Vestal. 1982. Effectsof petroleum hydrocarbons on plant litter microbiota in an arcticlake. Appl. Environ. Microbiol. 43:129-135.

25. McLean, E. 0. 1982. Soil pH and lime requirement, p. 199-234.In A. L. Page, R. H. Miller, and D. R. Keeney (ed.), Methodsof soil analysis, part 2. Chemical and microbiological properties.American Society of Agronomy, Inc., Soil Science Society ofAmerica, Inc., Madison, Wis.

26. Mueller, J. G., P. J. Chapman, and P. H. Pritchard. 1989.Creosote contaminated soils: their potential for bioremediation.Environ. Sci. Technol. 23:1197-1201.

27. Mueller, J. G., S. E. Lantz, B. 0. Blattmann, and P. J.Chapman. 1991. Bench-scale evaluation of alternative biologicaltreatment processes for the remediation of pentachlorophenol-and creosote-contaminated materials: slurry-phase bioremedia-tion. Environ. Sci. Technol. 25:1055-1061.

28. Mueller, J. G., D. P. Middaugh, S. E. Lantz, and P. J. Chapman.1991. Biodegradation of creosote and pentachlorophenol incontaminated groundwater: chemical and biological assess-ment. Appl. Environ. Microbiol. 57:1277-1285.

29. Orvos, D. R., G. H. Lacy, and J. Cairns. 1990. Geneticallyengineered Erwinia carotovora: survival, intraspecific competi-tion, and effects upon selected bacterial genera. Appl. Environ.Microbiol. 56:1689-1694.

30. Park, K. S., R. C. Sims, R. R. Dupont, W. J. Doucette, and J. E.Matthews. 1990. Fate of PAH compounds in two soil types:

APPL. ENVIRON. MICROBIOL.

on March 25, 2020 by guest

http://aem.asm

.org/D

ownloaded from

MINERALIZATION OF PAHs AND NHAs IN SOIL 3469

influence of volatilization, abiotic loss and biological activity.Environ. Toxicol. Chem. 9:187-195.

31. Postma, J., and J. A. van Veen. 1990. Habitable pore space andsurvival of Rhizobium leguminosarum biovar trifolii introducedinto soil. Microb. Ecol. 19:149-161.

32. Robinson, J. A. 1985. Determining microbial kinetic parametersusing nonlinear regression analysis: advantages and limitationsin microbial ecology. Adv. Microb. Ecol. 8:61-114.

33. Rogoff, M. H. 1962. Chemistry of oxidation of polycyclicaromatic hydrocarbons by soil pseudomonads. J. Bacteriol.83:998-1004.

34. Sherrill, T. W., and G. S. Sayler. 1980. Phenanthrene biodegra-dation in freshwater environments. Appl. Environ. Microbiol.39:172-178.

35. Sims, R. C., and M. R. Overcash. 1983. Fate of polynucleararomatic compounds (PNAs) in soil-plant systems. ResidueRev. 88:1-68.

36. Skerman, V. B. D. 1967. A guide to the identification of thegenera of bacteria, 2nd ed. The Williams & Wilkins Co.,Baltimore.

37. Van Elsas, J. D., J. T. Trevors, L. S. Van Overbeek, and M. E.Starodub. 1989. Survival of Pseudomonas fluorescens contain-ing plasmids RP4 or pRK2501 and plasmid stability after intro-duction into two soils of different texture. Can. J. Microbiol.35:951-959.

38. Wang, X., X. Yu, and R. Bartha. 1990. Effect of bioremediationon polycyclic aromatic hydrocarbon residues in soil. Environ.Sci. Technol. 24:1086-1089.

39. Weissenfels, W. D., M. Beyer, and J. Klein. 1990. Degradation ofphenanthrene, fluorene and fluoranthene by pure bacterial cul-tures. Appl. Microbiol. Biotechnol. 32:479-484.

40. White, D. C., W. M. Davis, J. F. Nickels, J. D. King, and R. J.Bobbie. 1979. Determination of sedimentary microbial biomassby extractable lipid phosphate. Oecologia 40:51-62.

41. Wolium, A. G. 1982. Cultural methods for soil microorganisms,p. 781-802. In A. L. Page, R. H. Miller, and D. R. Keeney (ed.),Methods of soil analysis, part 2. Chemical and microbiologicalproperties. American Society of Agronomy, Inc., Soil ScienceSociety of America, Inc., Madison, Wis.

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